Joyce C M
Department of Molecular Biophysics and Biochemistry, Yale University Medical School, New Haven, Connecticut 06510.
J Biol Chem. 1989 Jun 25;264(18):10858-66.
The polymerase and 3'-5'-exonuclease activities of the Klenow fragment of DNA polymerase I are located on separate structural domains of the protein, separated by about 30 A. To determine whether a DNA primer terminus can move from one active site to the other without dissociation of the enzyme-DNA complex, we carried out reactions on a labeled DNA substrate in the presence of a large excess of unlabeled DNA, to limit observations to a single enzyme-DNA encounter. The results indicated that while Klenow fragment is capable of intramolecular shuttling of a DNA substrate between the two catalytic sites, the intermolecular pathway involving enzyme-DNA dissociation can also be used. Thus, there is nothing in the protein structure or the reaction mechanism that dictates a particular means of moving the DNA substrate. Instead, the use of the intermolecular or the intramolecular pathway is determined by the competition between the polymerase or exonuclease reaction and DNA dissociation. When the substrate has a mispaired primer terminus, DNA dissociation seems generally more rapid than exonucleolytic digestion. Thus, Klenow fragment edits its own polymerase errors by a predominantly intermolecular process, involving dissociation of the enzyme-DNA complex and reassociation of the DNA with the exonuclease site of a second molecule of Klenow fragment.
DNA聚合酶I的Klenow片段的聚合酶活性和3'-5'-核酸外切酶活性位于该蛋白质的不同结构域上，两者相距约30埃。为了确定DNA引物末端能否在不解离酶-DNA复合物的情况下从一个活性位点移动到另一个活性位点，我们在大量未标记DNA存在的情况下对标记的DNA底物进行反应，以便将观察限制在单个酶-DNA相遇事件中。结果表明，虽然Klenow片段能够使DNA底物在两个催化位点之间进行分子内穿梭，但涉及酶-DNA解离的分子间途径也可能被采用。因此，蛋白质结构或反应机制中没有任何因素决定移动DNA底物的特定方式。相反，分子间或分子内途径的使用取决于聚合酶或核酸外切酶反应与DNA解离之间的竞争。当底物的引物末端存在错配时，DNA解离通常比核酸外切酶消化更快。因此，Klenow片段主要通过分子间过程来校正自身的聚合酶错误，该过程涉及酶-DNA复合物的解离以及DNA与第二个Klenow片段分子的核酸外切酶位点重新结合。
